When insulin binds to the insulin receptor, this stimulates endocytosis of the hormone-receptor complex. Ligand-stimulated endocytosis requires that the receptor must possess intact tyrosine kinase activity. In this project, we have demonstrated that the dileucine motif (EKITLL, amino acids 982-987) found in the juxtamembrane domain of the insulin receptor functions as sorting signals in the intracellular trafficking of the insulin receptor. Substitution of alanine residues for L986 and L987 in the juxtamembrane dileucine motif led to an 80% reduction in the rate of endocytosis. Nevertheless, the dileucine sequence is not conserved in the type 1 IGF receptor (i.e., EKITMS substituted for EKITLL). Accordingly, we constructed a double mutant insulin receptor containing the sequence of the IGF receptor (L986M/L987S), but the L986M/L987S-mutation did not impair endocytosis of the insulin receptor. This raised the question of whether MS could be substituted for LL in this dileucine motif. To address this question, we utilized the original assay for dileucine motifs by constructing a chimeric molecule in which the Tac antigen was fused to the EKITMS mutant sequence. In contrast to the observations with the mutant insulin receptor, the EKITMS sequence did not fully mimic the activity of the dileucine motif in targeting the mutant Tac fusion protein to lysosomes. It is not clear why the two experimental approaches led to somewhat different conclusions. However, it seems likely that there may be multiple intracellular "receptors" for dileucine motifs, with distinct "receptors" mediating different functions and exhibiting different binding specificities. In a different set of experiments, we have attempted to identify proteins that interact with cytoplasmic domains of receptor tyrosine kinases, and target them for endocytosis. Toward that end, we have investigated the protein sortin nexin 1 (Snx1), a protein that binds to the EGF receptor and appears to target that receptor for endocytosis. Specifically, we have characterized the binding specificity of Snx1 by assaying its ability to bind to other receptor tyrosine kinases. In addition, we have identified five additional cDNA encoding five other sorting nexins.